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An autoradiographic study of neurotensin receptors in the human hypothalamus
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An autoradiographic study of neurotensin receptors in the human hypothalamus Mohamed Najimi a,∗ , Alain Sarrieau b , Nicolas Kopp c , Fatiha Chigr c a b c
Laboratoire Génie Biologique, Faculty of Sciences and Techniques, Sultan Moulay Slimane University, P.O. Box: 523, 23000 Beni Mellal, Morocco EA 2972 Régulations Neuroendocriniennes, Avenue des Facultés, 33405 Talence Cedex, France Hôpital Neurologique et Neurochirurgical Pierre Wertheimer, 59, Boulevard Pinel, 69500 Bron, France
a r t i c l e
i n f o
Article history: Received 23 June 2013 Received in revised form 6 September 2013 Accepted 8 September 2013 Available online xxx Keywords: Human Brain Neurotensin receptors Hypothalamus Autoradiography
a b s t r a c t The aim of the present investigation was to determine a detailed mapping of neurotensin (NT) in the human hypothalamus, the brain region involved in neuroendocrine control. For this, we investigated the presence and the distribution of neurotensin binding sites in the human hypothalamus, using an in vitro quantitative autoradiography technique and the selective radioligand monoiodo-Tyr3neurotensin (2000 Ci/mM). This study was performed on nine adult human postmortem hypothalami. We first determined the biochemical kinetics of the binding and found that binding affinity constants were of high affinity and do not differ significantly between all cases investigated. Our analysis of the autoradiographic distribution shows that NT binding sites are widely distributed throughout the rostrocaudal extent of the hypothalamus. However, the distribution of NT binding sites is not homogenous and regional variations exist. In general, the highest densities are mainly present in the anterior hypothalamic level, particularly in the preoptic region and the anterior boarding limit (i.e. the diagonal band of Broca). Important NT binding site densities are also present at the mediobasal hypothalamic level, particularly in the paraventricular, parafornical and dorsomedial nuclei. At the posterior level, relatively moderate densities could be observed in the mammillary complex subdivisions, apart from the supramammillary nucleus and the posterior hypothalamic area. In conclusion, the present study demonstrates the occurrence of high concentrations of NT binding sites in various structures in many regions in the human adult hypothalamus, involved in the control of neuroendocrine and/or neurovegetative functions. © 2013 Elsevier GmbH. All rights reserved.
Introduction Neurotensin (NT) is a tridecapeptide originally isolated from mammalian brain (Carraway and Leeman, 1973). It is mainly expressed in central nervous systems and gastrointestinal tracts of both mammals and non-mammalian vertebrates. Previous physiological data demonstrated that NT plays a role as neurotransmitter
Abbreviations: AA, anterior hypothalamic area; Ac, anterior commissure; CP, cerebral peduncle; DA, dorsal hypothalamic area; Dbh, diagonal band of Broca horizontalis; Dbv, diagonal band of Broca verticalis; DM, dorsomedial nucleus; F, fornix; I, infundibular nucleus; L, lamina terminalis; LH, lateral hypothalamic area; lM, lateral mammillary nucleus; LP, lateral preoptic area; Lt, lateral mammillary nucleus; Me, median eminence; MM, medial mammillary nucleus; MP, medial preoptic area; Mt, medial tuberal nucleus; Oc, optic chiasma; On, optic nerve; Ot, optic tract; PA, posterior hypothalamic area; Pe, periventricular nucleus; P, premammillary nucleus; pM, paramammillary nucleus; Pv, paraventricular nucleus; Sb, subthalamic nucleus; SM, supramammillary nucleus; So, supraoptic nucleus; St, septothalamic nucleus; TCA, tubero cinereum area; Tm, tuberomammillary nucleus; Vm, ventromedial nucleus; Z, zona incerta; 3V, third ventricle. ∗ Corresponding author: Tel.: +212 523421141; fax: +212 523485201. E-mail addresses: [email protected], [email protected] (M. Najimi).
or neuromodulator in the central nervous system (Pan et al., 1992; Rostène and Alexander, 1997; Katsanos et al., 2008). Intracisternal injections of NT in rats have been shown to lower body temperature (Gordon et al., 2003), decrease food intake (Brunetti et al., 2005; Kalafatakis and Triantafyllou, 2011), reduce locomotor activity (Boules et al., 2001; Liang et al., 2010) and to produce an analgesic effect (Dobner, 2005). Detection of this neuropeptide in the hypothalamus has been related to neuroendocrine control of pituitary hormone secretions (Vijayan et al., 1994; Hentschel et al., 1998; Sicard et al., 2005; Kempadoo et al., 2013). The involvement of NTergic neurons in the regulation of neuroendocrine secretion has been supported by numerous observations. High NT endogenous levels have been detected in the hypothalamic region of both the rat and human as demonstrated by radioimmunoassay (Uhl and Snyder, 1976; Cooper et al., 1981; Manberg et al., 1982). NT-immunoreactive (IR) cell bodies have also been seen in many hypothalamic structures such as the paraventricular and infundibular nuclei, which send their fibers to the median eminence (Merchenthaler and Lennard, 1991). Intracerebroventricular or local NT injections in some hypothalamic areas have been associated with dramatic alterations of the plasma levels of most anterior
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2 Table 1 Source of brain tissues. Cases
Sex
Age (y)
Post-mortem delay (h)
Cause of death
A B C D E F G H I
M F M F F M M F F
22 27 34 42 45 47 67 82 82
30 25 28.5 12 20 12 7 2.5 34
Cardiac failure Cardiac failure Sudden death Sudden death Coronary thrombosis Cardiac failure Myocardial infarctus Cardiac failure Cardiac failure
pituitary hormones (Alexander and Leeman, 1992; Vijayan et al., 1994; Rostène and Alexander, 1997; Sicard et al., 2005). In the human CNS, detailed topographical organization of NT binding sites or receptors has only been documented for the spinal cord (Faull et al., 1989a), cortical and subcortical areas (Faull et al., 1989b; Quirion et al., 1982, 1992) and the medulla oblongata of the brainstem (Chigr et al., 1992). Moreover, these studies have not reported the presence of two NT receptor types as shown in the rat (Rostène and Alexander, 1997). To the best of our knowledge, no study has yet focused on the detailed distribution of these neuropeptide binding sites in the hypothalamic region despite the important role of NT in neuroendocrine functions. In the current study, we provide a detailed distribution of NT binding sites in the rostrocaudal extent of the human adult hypothalamus. Materials and methods
washed with ice cold buffer four times for two minutes each and rapidly dried with cold air. Labeled sections and iodinated standards (Amersham, Courtaboeuf Cedex, France) were then apposed to 3H sensitive Ultrofilm (Amersham) in Amersham exposure cassettes. After 2 weeks exposure in dark conditions at 4 ◦ C, the film was developed in Kodak D19 (Eastman Kodak, Rochester, NY, USA) for 3 min, dipped in water and fixed with Kodak rapid fixer for 10 min. Competition experiments were performed by incubating serial sections from the anterior and mediobasal hypothalamic levels in the same medium containing graded concentrations of unlabelled NT (10−12 to 10−6 M). IC50 values were calculated from inhibition curves as peptide concentrations inhibiting 50% of monoiodo 125 I-Tyr-neurotensin binding. Kinetics (IC 50 and KD ) analysis was computed by the method of Parker and Waud (1971). Densitometric analysis of 125 I-NT binding was carried out according to the methods described previously (Chigr et al., 1992) using a computer assisted image analysis system (Biocom 2000, les Ulis, France) by means of the standards coexposed. Values for total and non-specific binding of 125 I-NT were obtained for each region by averaging four to eight readings for each hypothalamic structure on an individual autoradiograph. Results were expressed as means ± S.E.M. Results Computer analysis of binding isotherms (Fig. 1A and B) showed that the apparent constant Kd and IC50 are respectively in the range of 0.31–0.42 nM and 0.61–0.75 nM.
Source and preparation of human tissues
Autoradiographic localization of 125 I-NT binding
We studied neurotensin binding sites in the hypothalami of nine adults, autopsied at the Edouard Herriot and Lyon Sud Hospitals (Lyon, France). None of these subjects died as a result of neurologic, psychiatric or endocrine disease (Table 1) and no pathological lesions were observed after macroscopic and microscopic examination of the brains. The study was approved by the Ethics Committee of the two French laboratories. At autopsy, the brains were removed from the cranium and the hypothalami dissected out at 4 ◦ C taking the parallel plane joining the optic chiasma and the anterior commissure as frontal plane and the caudal plane just behind the mammillary complex. The hypothalami were immediately frozen at −80 ◦ C and stored at the same temperature until mounted on cryostat chucks. The frozen hypothalami were then sliced as 20 m thick coronal sections at −20 ◦ C (Frigocut 2800, Reichert Jung, Heidelberg, Germany). Sections were collected onto chrome alum gelatin coated slides (Mentzel-Gläser, Braunschweig, Germany) and stored at −20 ◦ C until use. For the anatomical localization of the hypothalamic nuclei and areas, adjacent sections to those used for autoradiography were stained with cresyl violet. The identification was made according to the cytoarchitectonal drawings of the human hypothalamus of Braak and Braak (1987) and Diepen (1962).
The autoradiographic labeling is present throughout the rostrocaudal extent of the hypothalamic region. Across all hypothalamic levels and structures analyzed, the non-specific binding, as determined in the presence of 1 M NT1–13 was small throughout the concentration range (<11% of total binding). The distribution of NT binding sites throughout the human adult hypothalamus is shown in Figs. 1–6 and quantification of 125 I-NT binding site densities in the different hypothalamic structures is presented in Table 2. The detailed mapping has been realized in respect to the three anatomical hypothalamic levels i.e. anterior, mediobasal and posterior hypothalamus.
In vitro quantitative autoradiography Before autoradiography processing, the slides of the hypothalamus were warmed to room temperature. They were then incubated at 4 ◦ C for 2 h, with 0.1 nM monoiodo 125 I-Tyr-neurotensin (2000 Ci/mmol) in 50 mM Tris–HCl buffer (pH: 7.5) containing 5 mM MgCl2 , 0.2% bovine serum albumin and 0.02 mM bacitracin (New England Nuclear: NEN, Wellesley, MA, USA). Non-specific binding was determined as the binding of 125 I-NT in the presence of 1 M of unlabelled NT1-13 . After incubation, the slides were
Anterior hypothalamus A prominent feature of this hypothalamic level was the presence of an intense autoradiographic binding signal in the diagonal band of Broca (Fig. 2A). This binding outlined the entire structure, both in the vertical and horizontal limbs, and was evenly distributed in the two portions. However, this binding tends to decrease at the caudal level of the structure, particularly in the horizontal limb. More caudally, the medial and lateral preoptic areas showed also an intense binding (Figs. 2B and 3A). Similarly, the density of NT binding sites tended to decrease antero-posteriorly. The suprachiasmatic nucleus (Fig. 3B) presented a diffuse distribution of moderate binding densities throughout the structure contrasting with the low binding present in the anterior hypothalamic area (Figs. 3C, D and 4A). At this level, the dorsal and lateral hypothalamic areas as well as the organum vasculosum of lamina terminalis (Figs. 3C, D and 4A) also show very low autoradiographic labeling equivalent to that of the anterior hypothalamic area. Dorsally, low to moderate autoradiographic labeling was revealed in the septo-hypothalamic nucleus, whereas the nucleus of the stria terminalis displayed a dense binding (Fig. 3B–D). At the median
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Fig. 2. Autoradiographic images of neurotensin binding sites in coronal hypothalamic sections obtained in human adult. This figure represents the most rostral level of the anterior hypothalamus. A represents the edge of the rostral level at the diagonal band of Broca (septo-hypothalamic junction), and B represents the beginning of the hypothalamus senso stricto (preoptic area level). Scale bar = 3.5 mm.
Table 2 Densities of [125 I]-neurotensin1–13 in different human adult hypothalamic structures at the three levels. The densities of binding sites are expressed as mean ± S.E.M. of femtomoles of specifically bound per mg of protein (fmole/mg protein S.E.M.) of adults. Hypothalamic structures
Anterior hypothalamus Diagonal band of Broca Horizontal limb Vertical limb Medial preoptic area Lateral preoptic area Bed nucleus stria terminalis Septohypothalamic nucleus Organum vasculosum of the Lamina terminalis Supraoptic nucleus Suprachiasmatic nucleus Priventricular nucleus Anterior hypothalamic area Dorsal hypothalamic area Lateral hypothalamic area
Fig. 1. A – Scatchard plot representation of saturation curve; B: Bound and F: Free. B – Inhibition of specific 125 I-NT binding to hypothalamic sections by unlabeled NT.
level, low densities of NT binding sites were detected in the periventricular and paraventricular nuclei (Fig. 4A and B). The supraoptic nucleus presented a relatively moderate autoradiographic binding, principally in the ventrolateral part (Figs. 3C, D and 4A, B). Finally, the fornix column which emerged at this level as well as the anterior commissure, showed no binding (Figs. 2–4). Mediobasal hypothalamus Generally, this hypothalamic level presented relatively low autoradiographic labeling throughout its whole extent, particularly in the ventral portion. The infundibular, the ventromedial and the dorsomedial nuclei displayed equivalent low pattern of labeling
[125 I]-neurotensin1–13 specific binding (fmol/mg prot)
25.3 22.4 12.3 11.4 16.4 7.3 3.4 8.4 10.4 5.4 4.8 4.9 5.1
± ± ± ± ± ± ± ± ± ± ± ± ±
2.3 2.2 1.5 1.1 1.8 0.8 0.4 0.6 1.1 0.5 0.4 0.5 0.6
Mediobasal hypothalamus Paraventricular nucleus Anterior part Posterior part Infundibulum Tuber cinereum area Ventromedial nucleus Dorsomedial nucleus Medial tuberal nucleus Lateral tuberal nucleus Tuberomammillary nucleus Premammillary nucleus
4.1 7.5 2.6 4.1 5.1 5.3 4.6 4.1 5.3 2.1
± ± ± ± ± ± ± ± ± ±
0.3 0.7 0.5 0.3 0.6 0.4 0.4 0.3 0.4 0.2
Posterior hypothalamus Medial mammillary nucleus Lateral mammillary nucleus Intercalatus nucleus Supramammillary nucleus Paramammillary nucleus Submammillary nucleus Zona incerta
5.6 8.4 7.4 14.3 7.9 5.6 6.9
± ± ± ± ± ± ±
0.8 0.6 0.4 1.4 0.6 0.8 0.6
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Fig. 3. Regional distribution of NT binding sites as labeled by [125 I]-neurotensin1–13 , in the adult human hypothalamus, at the anterior level. These series of autoradiographic images illustrate the rostrocaudal distribution of NT binding sites at this hypothalamic level. Note that at rostral level, the binding is higher in the preoptic region, whereas it decreases caudally at the anterior and dorsal areas. Scale bar = 4 mm.
(Figs. 4 and 5A–C). In contrast to the anterior hypothalamic level, moderate NT binding site densities were present in the mediobasal part of the paraventricular nucleus as well as in the periventricular nucleus (Fig. 5). In the lateral part of this studied area, the parafornical nucleus also displayed moderate densities. In the posterior hypothalamic area, NT binding site density was moderate, existing
principally in both its ventral and medial parts. The tuberal nuclei displayed low to moderate NT binding site density (Figs. 5D and 6A), the autoradiographic labeling being homogenously present in both the medial and lateral portions. At the mediobasal hypothalamic level, the labeling present in the lateral hypothalamic area tended to relatively increase but remained in the low density average. Finally, no significant labeling was present in the median eminence. Posterior hypothalamus In the transition zone between the mediobasal and the posterior hypothalamus, only low but significant NT binding site densities were detected in the premammillary nucleus and the surrounding areas (Figs. 5B and 6A). At the posterior level, the mammillary complex presented a diffuse and heterogenous distribution of NT binding (Fig. 6A) with a low labeling in the medial mammillary nucleus (Fig. 6B). This binding was homogenously present in all the subdivisions of the nucleus. Inversely, the lateral mammillary nucleus was more labeled and presented moderate NT binding densities (Fig. 6B). The binding was principally detected in the ventral portion and was equivalent to the one revealed in the intercalatus nucleus (Fig. 5B). Dorsally, the autoradiographic labeling analysis reveals the highest NT binding sites in the supramammillary nucleus as also observed in the posterior hypothalamic area (Fig. 6B). Laterally to these structures, moderate NT binding site density was present in the subthalamicus nucleus and the zona incerta (Fig. 6B), but some structures, such as the mamillothalamic tract and the Vicq d’Azyr fibers tract surrounding the medial mammillary nucleus were devoid of binding. Discussion
Fig. 4. Detailed autoradiographic distribution of NT binding sites in the adult human mediobasal hypothalamic level. A represents the junction between the anterior and the mediobasal levels. B represents coronal section obtained more caudally. Scale bar = 3.5 mm.
The current study is the first, to the best of our knowledge, to provide a fine and detailed analysis of the anatomical distribution and quantification of NT binding sites in the human adult hypothalamus. While several studies have reported the presence of NT binding sites in the human CNS, none of them has focused on this brain region involved in vegetative and neuroendocrine functions.
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Fig. 5. Microphotographs obtained at the mediobasal level (A–C) and posterior level (D), in human adult hypothalamus, illustrating the rostrocaudal variations of NT binding sites. Scale bar = 4 mm.
In a previous study, analyzing the overall distribution of NT binding sites in the whole human brain (Sarrieau et al., 1985), their presence has been mentioned in the hypothalamic region without providing detailed mapping of their distribution. In our previous autoradiographic studies, we have demonstrated that monoiodo[125 I]Tyr3 neurotensin1–13 specifically binds with high affinity to sites on the human brain (Chigr et al., 1992). Moreover, competition curves indicate the presence of a single apparent population of NT binding sites under our experimental conditions, as already reported in rats (Moyse et al., 1987) and human (Sarrieau et al., 1985; Chigr et al., 1992). The analysis of NT binding sites, as demonstrated by in vitro quantitative autoradiography throughout the human hypothalamus, showed that their distribution was heterogeneous with high amounts of specific binding in the medial part of the three hypothalamic levels (anterior, mediobasal and posterior parts). High and relatively high densities were found in the diagonal band of Broca, the preoptic region and the bed nucleus of the stria terminalis. The paraventricular (mediobasal portion), the suprachiasmatic and the parafornical nuclei were moderately labeled, while only low to very low densities of binding sites were found in the lateral, dorsal and anterior hypothalamic areas as well as the periventricular and medial mammillary nuclei. In rat, it has been shown that some hypothalamic structures such as diagonal band of Broca, suprachiasmatic, dorsomedial, mammillary nuclei, display both NT receptor mRNA and high resolution of 125 I-NT binding. These areas demonstrated a selective association of NT binding sites with neuronal cell bodies. In contrast, in the other structures analyzed (i.e. supraoptic, paraventricular, ventromedial and infundibular nuclei, bed nucleus of the stria terminalis and anterior hypothalamic area), the presence of NT binding sites was not correlated to that of NT receptor mRNA expression, suggesting that NT receptors were mainly located on axon terminals. In this study, the distribution of NT binding sites has concerned different cases with different postmortem delays (3–38 h). No correlation was found between the anatomical distribution binding
sites density with such a parameter, which is in accordance with previous autoradiographic studies concerning other neurotransmitter or peptide binding sites in the postmortem human brain (Sarrieau et al., 1985; Chigr et al., 1992; Najimi et al., 2001, 2006). Furthermore, we found no significant correlation between gender and the anatomical distribution. Compared to rat hypothalamus, NT binding sites in the human hypothalamus, exhibit similar distribution and density profiles (Moyse et al., 1987). Some structures such as the supramammillary and the suprachiasmatic nuclei show high density of NT binding sites in contrast to dorsomedial and ventromedial hypothalamic nuclei, as shown both in rat and human. However, some differences should be highlighted such as lowly labeled paraventricular nucleus (anterior hypothalamic level) in human, but expressing high NT binding sites density in rat (Emson et al., 1990; Nicot et al., 1994). Inversely, the rat medial and lateral preoptic areas did not reveal any NT binding sites, whereas these structures, especially the medial preoptic area, are enriched with NT binding sites in the human brain. Finally, we also report the presence of relatively low density of NT binding sites in the human supraoptic nucleus, whereas it is devoid of such autoradiographic labeling in the rat (Young and Kuhar, 1981; Quirion et al., 1982). These discrepancies may reflect species differences. These variations seem phylogenetically dependent since the radioligand and experimental conditions used in these studies are similar to those used in the present work. It is noteworthy that some structures such as the parafornical, tuberal and paramammillary nuclei reported to be labeled here, have not been analyzed, as far as we are aware, in rat. As we reported earlier, some of these structures, in human do not correspond anatomically to those denominated in rat (Najimi et al., 1991). This is well illustrated for the tuberal nuclei, which are well differentiated only in primates (Bleir et al., 1981) and with high degree in humans (Le Gros Clark, 1936). It is interesting to compare the distribution of NT binding sites with that of endogenous NT as revealed by immunohistochemistry. Despite the fact that these immunohistochemical data concern only
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Fig. 6. NT binding sites distribution in the posterior level of adult human hypothalamus. Microphotograph A represents the beginning of this hypothalamic level and microphotograph B, illustrates the distribution of NT radiolabelling in the mammillary complex. Note that all the components of the mammillary body display moderate to relatively higher auto-radiographic labeling except the medial mammillary nucleus. Scale bar = 3.5 mm.
several hypothalamic structures (Mai et al., 1987), the comparison shows that generally there is a good correlation between the endogenous peptide and the corresponding binding sites, notably in the anterior and mediobasal hypothalamic levels. However, this is not confirmed for several other structures such as the diagonal band of Broca displaying only low NT immunoreactivity but the high NT binding densities. On the other hand, in the posterior hypothalamic level, the medial mammillary nucleus containing the greatest of NT-immunoreactive terminals and fibers, displays only low levels of NT binding sites. The highest contrast is observed in the fornix column and the median eminence, two structures not presenting NT binding sites, despite the presence of a dense network of NT-IR fibers. For the fornix, this mismatch is explained by the fact that NT-IR fibers represent fibers of passage deriving from the subiculum as demonstrated using tracing methods (Chronister and Sikes, 1975). This last structure sends most of its fibers throughout the fimbria into the column of the fornix to terminate in the many hypothalamic structures such as mammillary complex. Concerning the median eminence, it has been reported that the fibers or nerve terminals originating from other hypothalamic structures such as paraventricular and infundibular nuclei seem to reach portal vessels to allow NT release in the hypothalamohypophysiotropic system (Emson et al., 1990). This could explain the mismatch observed and support the neuroendocrine role of the peptide (Rostène and Alexander, 1997). Thus, it is relevant to consider the findings of the present investigations in the human hypothalamus in the context of NT neuroendocrine functions. Indeed, NT plays an important interactive role in all components of the hypothalamic-anterior pituitary circuit, which is mediated by an endocrine, paracrine or/and autocrine manner, toward most of the anatomical regions that define this circuit. Interestingly, higher densities of NT binding sites are present in the preoptic region (medial and lateral parts). This region, principally the medial part,
is widely accepted as the regulatory center for luteinizing hormone (LH) secretion in female rats (Barraclough, 1994). The medial preoptic area (MPA) is also known to contain high concentrations of the hypophyseotropic factor responsible for the release of LH, LHRH (luteinizing hormone releasing hormone) (Najimi et al., 1990). This is in favor of the presence NT and LHR interactions for the control of the release of LH (Alexander and Leeman, 1992). This suggestion is supported by experimental studies showing that microinjection of NT in the MPA increased the magnitude of the circadian LH secretion in rat (Ferris et al., 1984). Furthermore, the use of NT anti-serum to immunoneutralize endogenous NT, resulted in the reduction in the magnitude of the LH surge (Alexander et al., 1989). The present anatomical data are consistent with the view that NT present in the preoptic area could mediate the stimulatory effects of estrogen on LHRH secretion (Alexander and Leeman, 1992). The preoptic region, which is also implicated in the control of other functions as thermoregulation (Briese, 1998), contains high amounts of endogenous peptide (Mai et al., 1987) and NT binding sites as shown in the present study, suggesting its involvement in the control of central temperature. This is supported by pharmacological studies showing that intracerebral injection of NT or its analogs, significantly influence central temperature, by causing hypothermia (Gordon et al., 2003). It has been reported that the most neuronal neurotensinergic system is in a close relation with catecholaminergic system, especially the dopaminergic system (Geisler et al., 2006). Effects of NT on dopaminergic transmission are more widespread than previously reported in all major dopamine (DA) pathways affected by NT, including those present in the hypothalamus (Rostène and Alexander, 1997). In rat, the DAergic group associated with NT binding sites is present in the arcuate nucleus (equivalent to infundibular nucleus in human). This represents the A12 DA neurons known to project into the median eminence and posterior lobe of the pituitary (Emson et al., 1990). Of interest, we reported in the current study, the presence of high densities of NT binding sites density in the tuberoinfundibular system as well as the paraventricular nucleus, two areas densely expressing DAergic cell groups. These findings are in accordance with a functional interaction between DA and NT in these structures, known to be a major site of for the integration of neuroendocrine functions. This could also represent an anatomical substrate of NT neuroendocrine actions that may be mediated via DA release (Berry and Gudelsky, 1992; Hentschel et al., 1998; Geisler et al., 2006). In rat, the hypothalamic DAergic system does not synthesize the high affinity NT receptor mRNA in contrast to mesencephalic DAergic neurons, suggesting a presynaptic action of NT through the activation of specific receptor located on nerve terminals (Nicot et al., 1994). The presence of NT binding sites in two hypothalamic structures highly involved in food intake regulation, namely the ventromedial and the paraventricular nuclei, strongly suggests that the neuropeptide modulates this function. Of interest, anatomical data demonstrated the presence of direct NT containing projections from these structures to the liver (Uyama et al., 2004). Additionally, physiological evidence indicates that NT plays an important role mainly in modulating intestinal function via cooperation with other hormones to establish a bridge between brain and gut for appetite, weight status and generally eating behavior adjustment (Schubert, 2008; Kalafatakis and Triantafyllou, 2011). In summary, NT binding sites are found at different sites of the hypothalamic region with different specific densities. It includes the major antero-posterior extent of the hypothalamus, but displays regional specificity. NT binding sites are present with high density in hypothalamic structures known to influence the release of pituitary hormones, suggesting the presence of interactions between NT and other hypothalamic neurotransmitters/neuropeptides for the regulation of the neuroendocrine
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system. Finally, the association of NT binding sites with other hypothalamic areas controlling non-endocrine processes, also suggests that NT may have multiple transmission and/or modulation complex roles in the human hypothalamus.
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An autoradiographic study of neurotensin receptors in the human hypothalamus Mohamed Najimi a,∗ , Alain Sarrieau b , Nicolas Kopp c , Fatiha Chigr c a b c
Laboratoire Génie Biologique, Faculty of Sciences and Techniques, Sultan Moulay Slimane University, P.O. Box: 523, 23000 Beni Mellal, Morocco EA 2972 Régulations Neuroendocriniennes, Avenue des Facultés, 33405 Talence Cedex, France Hôpital Neurologique et Neurochirurgical Pierre Wertheimer, 59, Boulevard Pinel, 69500 Bron, France
a r t i c l e
i n f o
Article history: Received 23 June 2013 Received in revised form 6 September 2013 Accepted 8 September 2013 Available online xxx Keywords: Human Brain Neurotensin receptors Hypothalamus Autoradiography
a b s t r a c t The aim of the present investigation was to determine a detailed mapping of neurotensin (NT) in the human hypothalamus, the brain region involved in neuroendocrine control. For this, we investigated the presence and the distribution of neurotensin binding sites in the human hypothalamus, using an in vitro quantitative autoradiography technique and the selective radioligand monoiodo-Tyr3neurotensin (2000 Ci/mM). This study was performed on nine adult human postmortem hypothalami. We first determined the biochemical kinetics of the binding and found that binding affinity constants were of high affinity and do not differ significantly between all cases investigated. Our analysis of the autoradiographic distribution shows that NT binding sites are widely distributed throughout the rostrocaudal extent of the hypothalamus. However, the distribution of NT binding sites is not homogenous and regional variations exist. In general, the highest densities are mainly present in the anterior hypothalamic level, particularly in the preoptic region and the anterior boarding limit (i.e. the diagonal band of Broca). Important NT binding site densities are also present at the mediobasal hypothalamic level, particularly in the paraventricular, parafornical and dorsomedial nuclei. At the posterior level, relatively moderate densities could be observed in the mammillary complex subdivisions, apart from the supramammillary nucleus and the posterior hypothalamic area. In conclusion, the present study demonstrates the occurrence of high concentrations of NT binding sites in various structures in many regions in the human adult hypothalamus, involved in the control of neuroendocrine and/or neurovegetative functions. © 2013 Elsevier GmbH. All rights reserved.
Introduction Neurotensin (NT) is a tridecapeptide originally isolated from mammalian brain (Carraway and Leeman, 1973). It is mainly expressed in central nervous systems and gastrointestinal tracts of both mammals and non-mammalian vertebrates. Previous physiological data demonstrated that NT plays a role as neurotransmitter
Abbreviations: AA, anterior hypothalamic area; Ac, anterior commissure; CP, cerebral peduncle; DA, dorsal hypothalamic area; Dbh, diagonal band of Broca horizontalis; Dbv, diagonal band of Broca verticalis; DM, dorsomedial nucleus; F, fornix; I, infundibular nucleus; L, lamina terminalis; LH, lateral hypothalamic area; lM, lateral mammillary nucleus; LP, lateral preoptic area; Lt, lateral mammillary nucleus; Me, median eminence; MM, medial mammillary nucleus; MP, medial preoptic area; Mt, medial tuberal nucleus; Oc, optic chiasma; On, optic nerve; Ot, optic tract; PA, posterior hypothalamic area; Pe, periventricular nucleus; P, premammillary nucleus; pM, paramammillary nucleus; Pv, paraventricular nucleus; Sb, subthalamic nucleus; SM, supramammillary nucleus; So, supraoptic nucleus; St, septothalamic nucleus; TCA, tubero cinereum area; Tm, tuberomammillary nucleus; Vm, ventromedial nucleus; Z, zona incerta; 3V, third ventricle. ∗ Corresponding author: Tel.: +212 523421141; fax: +212 523485201. E-mail addresses: [email protected], [email protected] (M. Najimi).
or neuromodulator in the central nervous system (Pan et al., 1992; Rostène and Alexander, 1997; Katsanos et al., 2008). Intracisternal injections of NT in rats have been shown to lower body temperature (Gordon et al., 2003), decrease food intake (Brunetti et al., 2005; Kalafatakis and Triantafyllou, 2011), reduce locomotor activity (Boules et al., 2001; Liang et al., 2010) and to produce an analgesic effect (Dobner, 2005). Detection of this neuropeptide in the hypothalamus has been related to neuroendocrine control of pituitary hormone secretions (Vijayan et al., 1994; Hentschel et al., 1998; Sicard et al., 2005; Kempadoo et al., 2013). The involvement of NTergic neurons in the regulation of neuroendocrine secretion has been supported by numerous observations. High NT endogenous levels have been detected in the hypothalamic region of both the rat and human as demonstrated by radioimmunoassay (Uhl and Snyder, 1976; Cooper et al., 1981; Manberg et al., 1982). NT-immunoreactive (IR) cell bodies have also been seen in many hypothalamic structures such as the paraventricular and infundibular nuclei, which send their fibers to the median eminence (Merchenthaler and Lennard, 1991). Intracerebroventricular or local NT injections in some hypothalamic areas have been associated with dramatic alterations of the plasma levels of most anterior
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2 Table 1 Source of brain tissues. Cases
Sex
Age (y)
Post-mortem delay (h)
Cause of death
A B C D E F G H I
M F M F F M M F F
22 27 34 42 45 47 67 82 82
30 25 28.5 12 20 12 7 2.5 34
Cardiac failure Cardiac failure Sudden death Sudden death Coronary thrombosis Cardiac failure Myocardial infarctus Cardiac failure Cardiac failure
pituitary hormones (Alexander and Leeman, 1992; Vijayan et al., 1994; Rostène and Alexander, 1997; Sicard et al., 2005). In the human CNS, detailed topographical organization of NT binding sites or receptors has only been documented for the spinal cord (Faull et al., 1989a), cortical and subcortical areas (Faull et al., 1989b; Quirion et al., 1982, 1992) and the medulla oblongata of the brainstem (Chigr et al., 1992). Moreover, these studies have not reported the presence of two NT receptor types as shown in the rat (Rostène and Alexander, 1997). To the best of our knowledge, no study has yet focused on the detailed distribution of these neuropeptide binding sites in the hypothalamic region despite the important role of NT in neuroendocrine functions. In the current study, we provide a detailed distribution of NT binding sites in the rostrocaudal extent of the human adult hypothalamus. Materials and methods
washed with ice cold buffer four times for two minutes each and rapidly dried with cold air. Labeled sections and iodinated standards (Amersham, Courtaboeuf Cedex, France) were then apposed to 3H sensitive Ultrofilm (Amersham) in Amersham exposure cassettes. After 2 weeks exposure in dark conditions at 4 ◦ C, the film was developed in Kodak D19 (Eastman Kodak, Rochester, NY, USA) for 3 min, dipped in water and fixed with Kodak rapid fixer for 10 min. Competition experiments were performed by incubating serial sections from the anterior and mediobasal hypothalamic levels in the same medium containing graded concentrations of unlabelled NT (10−12 to 10−6 M). IC50 values were calculated from inhibition curves as peptide concentrations inhibiting 50% of monoiodo 125 I-Tyr-neurotensin binding. Kinetics (IC 50 and KD ) analysis was computed by the method of Parker and Waud (1971). Densitometric analysis of 125 I-NT binding was carried out according to the methods described previously (Chigr et al., 1992) using a computer assisted image analysis system (Biocom 2000, les Ulis, France) by means of the standards coexposed. Values for total and non-specific binding of 125 I-NT were obtained for each region by averaging four to eight readings for each hypothalamic structure on an individual autoradiograph. Results were expressed as means ± S.E.M. Results Computer analysis of binding isotherms (Fig. 1A and B) showed that the apparent constant Kd and IC50 are respectively in the range of 0.31–0.42 nM and 0.61–0.75 nM.
Source and preparation of human tissues
Autoradiographic localization of 125 I-NT binding
We studied neurotensin binding sites in the hypothalami of nine adults, autopsied at the Edouard Herriot and Lyon Sud Hospitals (Lyon, France). None of these subjects died as a result of neurologic, psychiatric or endocrine disease (Table 1) and no pathological lesions were observed after macroscopic and microscopic examination of the brains. The study was approved by the Ethics Committee of the two French laboratories. At autopsy, the brains were removed from the cranium and the hypothalami dissected out at 4 ◦ C taking the parallel plane joining the optic chiasma and the anterior commissure as frontal plane and the caudal plane just behind the mammillary complex. The hypothalami were immediately frozen at −80 ◦ C and stored at the same temperature until mounted on cryostat chucks. The frozen hypothalami were then sliced as 20 m thick coronal sections at −20 ◦ C (Frigocut 2800, Reichert Jung, Heidelberg, Germany). Sections were collected onto chrome alum gelatin coated slides (Mentzel-Gläser, Braunschweig, Germany) and stored at −20 ◦ C until use. For the anatomical localization of the hypothalamic nuclei and areas, adjacent sections to those used for autoradiography were stained with cresyl violet. The identification was made according to the cytoarchitectonal drawings of the human hypothalamus of Braak and Braak (1987) and Diepen (1962).
The autoradiographic labeling is present throughout the rostrocaudal extent of the hypothalamic region. Across all hypothalamic levels and structures analyzed, the non-specific binding, as determined in the presence of 1 M NT1–13 was small throughout the concentration range (<11% of total binding). The distribution of NT binding sites throughout the human adult hypothalamus is shown in Figs. 1–6 and quantification of 125 I-NT binding site densities in the different hypothalamic structures is presented in Table 2. The detailed mapping has been realized in respect to the three anatomical hypothalamic levels i.e. anterior, mediobasal and posterior hypothalamus.
In vitro quantitative autoradiography Before autoradiography processing, the slides of the hypothalamus were warmed to room temperature. They were then incubated at 4 ◦ C for 2 h, with 0.1 nM monoiodo 125 I-Tyr-neurotensin (2000 Ci/mmol) in 50 mM Tris–HCl buffer (pH: 7.5) containing 5 mM MgCl2 , 0.2% bovine serum albumin and 0.02 mM bacitracin (New England Nuclear: NEN, Wellesley, MA, USA). Non-specific binding was determined as the binding of 125 I-NT in the presence of 1 M of unlabelled NT1-13 . After incubation, the slides were
Anterior hypothalamus A prominent feature of this hypothalamic level was the presence of an intense autoradiographic binding signal in the diagonal band of Broca (Fig. 2A). This binding outlined the entire structure, both in the vertical and horizontal limbs, and was evenly distributed in the two portions. However, this binding tends to decrease at the caudal level of the structure, particularly in the horizontal limb. More caudally, the medial and lateral preoptic areas showed also an intense binding (Figs. 2B and 3A). Similarly, the density of NT binding sites tended to decrease antero-posteriorly. The suprachiasmatic nucleus (Fig. 3B) presented a diffuse distribution of moderate binding densities throughout the structure contrasting with the low binding present in the anterior hypothalamic area (Figs. 3C, D and 4A). At this level, the dorsal and lateral hypothalamic areas as well as the organum vasculosum of lamina terminalis (Figs. 3C, D and 4A) also show very low autoradiographic labeling equivalent to that of the anterior hypothalamic area. Dorsally, low to moderate autoradiographic labeling was revealed in the septo-hypothalamic nucleus, whereas the nucleus of the stria terminalis displayed a dense binding (Fig. 3B–D). At the median
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Fig. 2. Autoradiographic images of neurotensin binding sites in coronal hypothalamic sections obtained in human adult. This figure represents the most rostral level of the anterior hypothalamus. A represents the edge of the rostral level at the diagonal band of Broca (septo-hypothalamic junction), and B represents the beginning of the hypothalamus senso stricto (preoptic area level). Scale bar = 3.5 mm.
Table 2 Densities of [125 I]-neurotensin1–13 in different human adult hypothalamic structures at the three levels. The densities of binding sites are expressed as mean ± S.E.M. of femtomoles of specifically bound per mg of protein (fmole/mg protein S.E.M.) of adults. Hypothalamic structures
Anterior hypothalamus Diagonal band of Broca Horizontal limb Vertical limb Medial preoptic area Lateral preoptic area Bed nucleus stria terminalis Septohypothalamic nucleus Organum vasculosum of the Lamina terminalis Supraoptic nucleus Suprachiasmatic nucleus Priventricular nucleus Anterior hypothalamic area Dorsal hypothalamic area Lateral hypothalamic area
Fig. 1. A – Scatchard plot representation of saturation curve; B: Bound and F: Free. B – Inhibition of specific 125 I-NT binding to hypothalamic sections by unlabeled NT.
level, low densities of NT binding sites were detected in the periventricular and paraventricular nuclei (Fig. 4A and B). The supraoptic nucleus presented a relatively moderate autoradiographic binding, principally in the ventrolateral part (Figs. 3C, D and 4A, B). Finally, the fornix column which emerged at this level as well as the anterior commissure, showed no binding (Figs. 2–4). Mediobasal hypothalamus Generally, this hypothalamic level presented relatively low autoradiographic labeling throughout its whole extent, particularly in the ventral portion. The infundibular, the ventromedial and the dorsomedial nuclei displayed equivalent low pattern of labeling
[125 I]-neurotensin1–13 specific binding (fmol/mg prot)
25.3 22.4 12.3 11.4 16.4 7.3 3.4 8.4 10.4 5.4 4.8 4.9 5.1
± ± ± ± ± ± ± ± ± ± ± ± ±
2.3 2.2 1.5 1.1 1.8 0.8 0.4 0.6 1.1 0.5 0.4 0.5 0.6
Mediobasal hypothalamus Paraventricular nucleus Anterior part Posterior part Infundibulum Tuber cinereum area Ventromedial nucleus Dorsomedial nucleus Medial tuberal nucleus Lateral tuberal nucleus Tuberomammillary nucleus Premammillary nucleus
4.1 7.5 2.6 4.1 5.1 5.3 4.6 4.1 5.3 2.1
± ± ± ± ± ± ± ± ± ±
0.3 0.7 0.5 0.3 0.6 0.4 0.4 0.3 0.4 0.2
Posterior hypothalamus Medial mammillary nucleus Lateral mammillary nucleus Intercalatus nucleus Supramammillary nucleus Paramammillary nucleus Submammillary nucleus Zona incerta
5.6 8.4 7.4 14.3 7.9 5.6 6.9
± ± ± ± ± ± ±
0.8 0.6 0.4 1.4 0.6 0.8 0.6
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Fig. 3. Regional distribution of NT binding sites as labeled by [125 I]-neurotensin1–13 , in the adult human hypothalamus, at the anterior level. These series of autoradiographic images illustrate the rostrocaudal distribution of NT binding sites at this hypothalamic level. Note that at rostral level, the binding is higher in the preoptic region, whereas it decreases caudally at the anterior and dorsal areas. Scale bar = 4 mm.
(Figs. 4 and 5A–C). In contrast to the anterior hypothalamic level, moderate NT binding site densities were present in the mediobasal part of the paraventricular nucleus as well as in the periventricular nucleus (Fig. 5). In the lateral part of this studied area, the parafornical nucleus also displayed moderate densities. In the posterior hypothalamic area, NT binding site density was moderate, existing
principally in both its ventral and medial parts. The tuberal nuclei displayed low to moderate NT binding site density (Figs. 5D and 6A), the autoradiographic labeling being homogenously present in both the medial and lateral portions. At the mediobasal hypothalamic level, the labeling present in the lateral hypothalamic area tended to relatively increase but remained in the low density average. Finally, no significant labeling was present in the median eminence. Posterior hypothalamus In the transition zone between the mediobasal and the posterior hypothalamus, only low but significant NT binding site densities were detected in the premammillary nucleus and the surrounding areas (Figs. 5B and 6A). At the posterior level, the mammillary complex presented a diffuse and heterogenous distribution of NT binding (Fig. 6A) with a low labeling in the medial mammillary nucleus (Fig. 6B). This binding was homogenously present in all the subdivisions of the nucleus. Inversely, the lateral mammillary nucleus was more labeled and presented moderate NT binding densities (Fig. 6B). The binding was principally detected in the ventral portion and was equivalent to the one revealed in the intercalatus nucleus (Fig. 5B). Dorsally, the autoradiographic labeling analysis reveals the highest NT binding sites in the supramammillary nucleus as also observed in the posterior hypothalamic area (Fig. 6B). Laterally to these structures, moderate NT binding site density was present in the subthalamicus nucleus and the zona incerta (Fig. 6B), but some structures, such as the mamillothalamic tract and the Vicq d’Azyr fibers tract surrounding the medial mammillary nucleus were devoid of binding. Discussion
Fig. 4. Detailed autoradiographic distribution of NT binding sites in the adult human mediobasal hypothalamic level. A represents the junction between the anterior and the mediobasal levels. B represents coronal section obtained more caudally. Scale bar = 3.5 mm.
The current study is the first, to the best of our knowledge, to provide a fine and detailed analysis of the anatomical distribution and quantification of NT binding sites in the human adult hypothalamus. While several studies have reported the presence of NT binding sites in the human CNS, none of them has focused on this brain region involved in vegetative and neuroendocrine functions.
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Fig. 5. Microphotographs obtained at the mediobasal level (A–C) and posterior level (D), in human adult hypothalamus, illustrating the rostrocaudal variations of NT binding sites. Scale bar = 4 mm.
In a previous study, analyzing the overall distribution of NT binding sites in the whole human brain (Sarrieau et al., 1985), their presence has been mentioned in the hypothalamic region without providing detailed mapping of their distribution. In our previous autoradiographic studies, we have demonstrated that monoiodo[125 I]Tyr3 neurotensin1–13 specifically binds with high affinity to sites on the human brain (Chigr et al., 1992). Moreover, competition curves indicate the presence of a single apparent population of NT binding sites under our experimental conditions, as already reported in rats (Moyse et al., 1987) and human (Sarrieau et al., 1985; Chigr et al., 1992). The analysis of NT binding sites, as demonstrated by in vitro quantitative autoradiography throughout the human hypothalamus, showed that their distribution was heterogeneous with high amounts of specific binding in the medial part of the three hypothalamic levels (anterior, mediobasal and posterior parts). High and relatively high densities were found in the diagonal band of Broca, the preoptic region and the bed nucleus of the stria terminalis. The paraventricular (mediobasal portion), the suprachiasmatic and the parafornical nuclei were moderately labeled, while only low to very low densities of binding sites were found in the lateral, dorsal and anterior hypothalamic areas as well as the periventricular and medial mammillary nuclei. In rat, it has been shown that some hypothalamic structures such as diagonal band of Broca, suprachiasmatic, dorsomedial, mammillary nuclei, display both NT receptor mRNA and high resolution of 125 I-NT binding. These areas demonstrated a selective association of NT binding sites with neuronal cell bodies. In contrast, in the other structures analyzed (i.e. supraoptic, paraventricular, ventromedial and infundibular nuclei, bed nucleus of the stria terminalis and anterior hypothalamic area), the presence of NT binding sites was not correlated to that of NT receptor mRNA expression, suggesting that NT receptors were mainly located on axon terminals. In this study, the distribution of NT binding sites has concerned different cases with different postmortem delays (3–38 h). No correlation was found between the anatomical distribution binding
sites density with such a parameter, which is in accordance with previous autoradiographic studies concerning other neurotransmitter or peptide binding sites in the postmortem human brain (Sarrieau et al., 1985; Chigr et al., 1992; Najimi et al., 2001, 2006). Furthermore, we found no significant correlation between gender and the anatomical distribution. Compared to rat hypothalamus, NT binding sites in the human hypothalamus, exhibit similar distribution and density profiles (Moyse et al., 1987). Some structures such as the supramammillary and the suprachiasmatic nuclei show high density of NT binding sites in contrast to dorsomedial and ventromedial hypothalamic nuclei, as shown both in rat and human. However, some differences should be highlighted such as lowly labeled paraventricular nucleus (anterior hypothalamic level) in human, but expressing high NT binding sites density in rat (Emson et al., 1990; Nicot et al., 1994). Inversely, the rat medial and lateral preoptic areas did not reveal any NT binding sites, whereas these structures, especially the medial preoptic area, are enriched with NT binding sites in the human brain. Finally, we also report the presence of relatively low density of NT binding sites in the human supraoptic nucleus, whereas it is devoid of such autoradiographic labeling in the rat (Young and Kuhar, 1981; Quirion et al., 1982). These discrepancies may reflect species differences. These variations seem phylogenetically dependent since the radioligand and experimental conditions used in these studies are similar to those used in the present work. It is noteworthy that some structures such as the parafornical, tuberal and paramammillary nuclei reported to be labeled here, have not been analyzed, as far as we are aware, in rat. As we reported earlier, some of these structures, in human do not correspond anatomically to those denominated in rat (Najimi et al., 1991). This is well illustrated for the tuberal nuclei, which are well differentiated only in primates (Bleir et al., 1981) and with high degree in humans (Le Gros Clark, 1936). It is interesting to compare the distribution of NT binding sites with that of endogenous NT as revealed by immunohistochemistry. Despite the fact that these immunohistochemical data concern only
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Fig. 6. NT binding sites distribution in the posterior level of adult human hypothalamus. Microphotograph A represents the beginning of this hypothalamic level and microphotograph B, illustrates the distribution of NT radiolabelling in the mammillary complex. Note that all the components of the mammillary body display moderate to relatively higher auto-radiographic labeling except the medial mammillary nucleus. Scale bar = 3.5 mm.
several hypothalamic structures (Mai et al., 1987), the comparison shows that generally there is a good correlation between the endogenous peptide and the corresponding binding sites, notably in the anterior and mediobasal hypothalamic levels. However, this is not confirmed for several other structures such as the diagonal band of Broca displaying only low NT immunoreactivity but the high NT binding densities. On the other hand, in the posterior hypothalamic level, the medial mammillary nucleus containing the greatest of NT-immunoreactive terminals and fibers, displays only low levels of NT binding sites. The highest contrast is observed in the fornix column and the median eminence, two structures not presenting NT binding sites, despite the presence of a dense network of NT-IR fibers. For the fornix, this mismatch is explained by the fact that NT-IR fibers represent fibers of passage deriving from the subiculum as demonstrated using tracing methods (Chronister and Sikes, 1975). This last structure sends most of its fibers throughout the fimbria into the column of the fornix to terminate in the many hypothalamic structures such as mammillary complex. Concerning the median eminence, it has been reported that the fibers or nerve terminals originating from other hypothalamic structures such as paraventricular and infundibular nuclei seem to reach portal vessels to allow NT release in the hypothalamohypophysiotropic system (Emson et al., 1990). This could explain the mismatch observed and support the neuroendocrine role of the peptide (Rostène and Alexander, 1997). Thus, it is relevant to consider the findings of the present investigations in the human hypothalamus in the context of NT neuroendocrine functions. Indeed, NT plays an important interactive role in all components of the hypothalamic-anterior pituitary circuit, which is mediated by an endocrine, paracrine or/and autocrine manner, toward most of the anatomical regions that define this circuit. Interestingly, higher densities of NT binding sites are present in the preoptic region (medial and lateral parts). This region, principally the medial part,
is widely accepted as the regulatory center for luteinizing hormone (LH) secretion in female rats (Barraclough, 1994). The medial preoptic area (MPA) is also known to contain high concentrations of the hypophyseotropic factor responsible for the release of LH, LHRH (luteinizing hormone releasing hormone) (Najimi et al., 1990). This is in favor of the presence NT and LHR interactions for the control of the release of LH (Alexander and Leeman, 1992). This suggestion is supported by experimental studies showing that microinjection of NT in the MPA increased the magnitude of the circadian LH secretion in rat (Ferris et al., 1984). Furthermore, the use of NT anti-serum to immunoneutralize endogenous NT, resulted in the reduction in the magnitude of the LH surge (Alexander et al., 1989). The present anatomical data are consistent with the view that NT present in the preoptic area could mediate the stimulatory effects of estrogen on LHRH secretion (Alexander and Leeman, 1992). The preoptic region, which is also implicated in the control of other functions as thermoregulation (Briese, 1998), contains high amounts of endogenous peptide (Mai et al., 1987) and NT binding sites as shown in the present study, suggesting its involvement in the control of central temperature. This is supported by pharmacological studies showing that intracerebral injection of NT or its analogs, significantly influence central temperature, by causing hypothermia (Gordon et al., 2003). It has been reported that the most neuronal neurotensinergic system is in a close relation with catecholaminergic system, especially the dopaminergic system (Geisler et al., 2006). Effects of NT on dopaminergic transmission are more widespread than previously reported in all major dopamine (DA) pathways affected by NT, including those present in the hypothalamus (Rostène and Alexander, 1997). In rat, the DAergic group associated with NT binding sites is present in the arcuate nucleus (equivalent to infundibular nucleus in human). This represents the A12 DA neurons known to project into the median eminence and posterior lobe of the pituitary (Emson et al., 1990). Of interest, we reported in the current study, the presence of high densities of NT binding sites density in the tuberoinfundibular system as well as the paraventricular nucleus, two areas densely expressing DAergic cell groups. These findings are in accordance with a functional interaction between DA and NT in these structures, known to be a major site of for the integration of neuroendocrine functions. This could also represent an anatomical substrate of NT neuroendocrine actions that may be mediated via DA release (Berry and Gudelsky, 1992; Hentschel et al., 1998; Geisler et al., 2006). In rat, the hypothalamic DAergic system does not synthesize the high affinity NT receptor mRNA in contrast to mesencephalic DAergic neurons, suggesting a presynaptic action of NT through the activation of specific receptor located on nerve terminals (Nicot et al., 1994). The presence of NT binding sites in two hypothalamic structures highly involved in food intake regulation, namely the ventromedial and the paraventricular nuclei, strongly suggests that the neuropeptide modulates this function. Of interest, anatomical data demonstrated the presence of direct NT containing projections from these structures to the liver (Uyama et al., 2004). Additionally, physiological evidence indicates that NT plays an important role mainly in modulating intestinal function via cooperation with other hormones to establish a bridge between brain and gut for appetite, weight status and generally eating behavior adjustment (Schubert, 2008; Kalafatakis and Triantafyllou, 2011). In summary, NT binding sites are found at different sites of the hypothalamic region with different specific densities. It includes the major antero-posterior extent of the hypothalamus, but displays regional specificity. NT binding sites are present with high density in hypothalamic structures known to influence the release of pituitary hormones, suggesting the presence of interactions between NT and other hypothalamic neurotransmitters/neuropeptides for the regulation of the neuroendocrine
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system. Finally, the association of NT binding sites with other hypothalamic areas controlling non-endocrine processes, also suggests that NT may have multiple transmission and/or modulation complex roles in the human hypothalamus.
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